Within the eukaryotic cell nucleus, genetic information is organized in a highly conserved structural polymer, termed chromatin, which supports and controls the crucial functions of the genome. The chromatin template undergoes dynamic changes during many genetic processes. These include necessary structural reorganizations that occur during DNA replication and cell cycle progression, spatially and temporally coordinated gene expression, as well as DNA repair and recombination events. The fundamental repeating unit of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around an octamer of core histone proteins, H2A, H2B, H3, and H4. Linker histones of the H1 class associate with DNA between single nucleosomes, establishing a higher level of organization, the so-called ‘solenoid’ helical or zig-zag fibers (30 nm fibers). Chromatin architecture beyond the 30 nm fibers is less clear, but folding and unfolding of putative superstructures is thought to have a pronounced impact on genomic function and gene activity.
Core histone proteins are evolutionary conserved and consist mainly of flexible N-terminal tails protruding outward from the nucleosome, and globular C-terminal domains making up the nucleosome scaffold. Histones function as acceptors for a variety of post-translational modifications, including acetylation, methylation and ubiquitination of lysine (K) residues, phosphorylation of serine (S) and threonine (T) residues, and methylation of arginine (R) residues (FIG. 1A). The different histone modifications and the corresponding enzymatic systems that maintain them have been reviewed extensively in the recent literature (e.g. Zhang et al., “Transcription Regulation by Histone Methylation: Interplay Between Different Covalent Modifications of the Core Histone Tails,” Genes Dev 15:2343-2360 (2000); Kouzarides, “Histone Methylation in Transcriptional Control,” Curr Opin Genet Dev 12:198-209 (2002); Lachner et al., “The Many Faces of Histone Lysine Methylation,” Curr Opin Cell Biol 14:286-298 (2002); Berger, Histone Modifications in Transcriptional Regulation,” Curr Opin Genet Dev 12:142-148 (2002); and Eberharter et al., “Histone Acetylation: A Switch Between Repressive and Permissive Chromatin,” Second in Review Series on Chromatin Dynamics. EMBO Rep 3:224-229 (2002)). Combinations of post-translational marks on single histones, single nucleosomes and nucleosomal domains establish local and global patterns of chromatin modification that may specify unique downstream functions (Strahl et al., “The Language of Covalent Histone Modifications,” Nature 403:41-45 (2000); Turner, “Histone Acetylation and an Epigenetic Code,” Bioessays 22:836-845 (2000)). These patterns can be altered by multiple extracellular and intracellular stimuli, and chromatin itself has been proposed to serve as signaling platform and to function as a genomic integrator of various signaling pathways (Cheung et al., “Signaling to Chromatin Through Histone Modifications,” Cell 103:263-271 (2000)).
One major challenge in chromatin biology is connecting particular modifications with distinct biological functions and vice versa. One of the better-understood histone modifications in that aspect is histone acetylation. It is now generally accepted that hyperacetylated histones are mostly associated with activated genomic regions, at both local and global levels. In contrast, deacetylation (leading to hypoacetylation) mainly results in repression and silencing (Turner, “Histone Acetylation and an Epigenetic Code,” Bioessays 22:836-845 (2000) and Grunstein, “Histone Acetylation in Chromatin Structure and Transcription,” Nature 389:349-352 (1997)). Interestingly, histone methylation appears to have multiple effects on chromatin function in a system- and site-specific manner. Methylation of H3 on K9, for example, is largely associated with silencing and repression in many species. Methylation of H3 on K4, on the other hand, is most often associated with active or permissive chromatin regions. However, deletion of the H3-K4 HMT, Set1, in budding yeast causes defects in rDNA silencing (Briggs et al., “Histone H3 Lysine 4 Methylation is Mediated by Set1 and Required for Cell Growth and rDNA Silencing in Saccharomyces cerevisiae,” Genes Dev 15:3286-3295 (2001) and Bryk et al., “Evidence that Set1, a Factor Required for Methylation of Histone H3, Regulates rDNA Silencing in S. cerevisiae by a Sir2-independent Mechanism,” Curr Biol 12:165-170 (2002)). These findings raise the question of whether methylation of H3 on K4 is also involved in gene repression in this organism. Similarly, methylation of H3-K36 has been suggested to be involved in transcriptional repression (Strahl et al., “Set2 is a Nucleosomal Histone H3-selective Methyltransferase that Mediates Transcriptional Repression,” Mol Cell Biol 22:1298-1306 (2002)), but the corresponding modifying enzyme, Set2, has been found in complex with actively transcribing (or elongation engaged) RNA PolII (Li et al., “Association of the Histone Methyltransferase Set2 with RNA Polymerase II Plays a Role in Transcription Elongation,” J Biol Chem 14:14 (2002)). Along with the dual personality of the phosphorylation of H3 at S10, which has been implicated in transcriptional activation, but also mitotic chromosome condensation (Cheung et al., “Synergistic Coupling of Histone H3 Phosphorylation and Acetylation in Response to Epidermal Growth Factor Stimulation,” Mol Cell 5:905-915 (2000)), these results argue that single histone modifications may have distinct biological effects depending on their context.
The findings that a particular post-translational modification might mediate separate, and sometimes opposing, physiological processes led to the suggestion that multiple readouts of a certain covalent mark could be obtained by various combinations of different modifications in the same chromatin region (Strahl et al., “The Language of Covalent Histone Modifications,” Nature 403:41-45 (2000) and Jenuwein et al., “Translating the Histone Code,” Science 293:1074-1080 (2001)). Indeed, the use of antibodies that recognize such combinations of post-translational marks, and the more recent application of novel mass spectrometry approaches, have verified that particular sets of modifications might occur concomitantly on the same histone tail (Cheung et al., “Synergistic Coupling of Histone H3 Phosphorylation and Acetylation in Response to Epidermal Growth Factor Stimulation,” Mol Cell 5:905-915 (2000); Zhang et al., “Histone Acetylation and Deacetylation: Identification of Acetylation and Methylation Sites of HeLa Histone H4 by Mass Spectrometry,” Mol Cell Proteomics 1:500-508 (2002); and Zhang et al., “Identification of Acetylation and Methylation Sites of Histone H3 from Chicken Erythrocytes by High-Accuracy Matrix-Assisted Laser Desorption Ionization-Time-of-Flight, Matrix-Assisted Laser Desorption Ionization-Postsource Decay, and Nanoelectrospray Ionization Tandem Mass Spectrometry,” Anal Biochem 306:259-269 (2002)). While the field is far away from deciphering the specific modification patterns at the level of single histones, single nucleosomes, and nucleosomal domains, mounting evidence suggests that different histone modifications can influence or ‘communicate’ with each other on several levels.
An ever-growing number of modification sites on both histone-tail and non-tail domains have been identified (for reference see FIG. 1 and (Zhang et al., “Transcription Regulation by Histone Methylation: Interplay Between Different Covalent Modifications of the Core Histone Tails,” Genes Dev 15:2343-2360 (2000))). Whereas serine and threonine residues are well-known phospho-acceptor sites, lysine and arginine residues have multiple choices of post-translational modification possibilities (FIG. 1A). For example, lysine residues in histones can be modified by acetylation, monoubiquitination or mono-, di-, and tri-methylation. Similarly, arginines might be mono- or di-methylated (symmetric or asymmetric) (Zhang et al., “Transcription Regulation by Histone Methylation: Interplay Between Different Covalent Modifications of the Core Histone Tails,” Genes Dev 15:2343-2360 (2000) and Bannister et al., “Histone Methylation: Dynamic or Static?,” Cell 109:801-806 (2002)). While it remains unclear as to what extent, if at all, individual residues undergo ‘choices’ of modification, it is well documented that H3-K9 and H3-K14 can be either acetylated or (mono-, di-, tri-) methylated (Zhang et al., “Histone Acetylation and Deacetylation: Identification of Acetylation and Methylation Sites of HeLa Histone H4 by Mass Spectrometry,” Mol Cell Proteomics 1:500-508 (2002) and Zhang et al., “Identification of Acetylation and Methylation Sites of Histone H3 from Chicken Erythrocytes by High-Accuracy Matrix-Assisted Laser Desorption Ionization-Time-of-Flight, Matrix-Assisted Laser Desorption Ionization—Postsource Decay, and Nanoelectrospray Ionization Tandem Mass Spectrometry,” Anal Biochem 306:259-269 (2002)). Obviously, different marks on the same site cannot co-exist, and, therefore, they exclude each other. An acetyl group, for example, must be removed before a methyl group can be added and complexes that contain both, HDACs and HMTs, have now been identified (Czermin et al., “Physical and Functional Association of SU(VAR)3-9 and HDAC1 in Drosophila,” EMBO Rep 2:915-919 (2001); Vaute et al., “Functional and Physical Interaction Between the Histone Methyl Transferase Suv39H1 and Histone Deacetylases,” Nucleic Acids Res 30:475-481 (2002); and Zhang et al., “Association of Class II Histone Deacetylases with Heterochromatin Protein 1: Potential Role for Histone Methylation in Control of Muscle Differentiation,” Mol Cell Biol 22:7302-7312 (2002)). Genetic studies in Schizosaccharomyces pombe have further shown that the HDAC, Clr6, is necessary for methylation at H3-K9 by the Clr4 HMT to occur (Nakayama et al., “Role of Histone H3 Lysine 9 Methylation in Epigenetic Control of Heterochromatin Assembly,” Science 292:110-113 (2001)).
It seems obvious that different modifications of a particular site can have different readouts and biological functions. Nevertheless, it is now known that the exact state of methylation (i.e. mono-, di- and tri-methylation) of a single lysine residue has an impact on physiological processes. For example, it was recently shown, that di-methylation of H3-K4 occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes (Santos-Rosa et al., “Active Genes are Tri-methylated at K4 of Histone H3,” Nature 419:407-411 (2002)). Similar studies investigating other sites of methylation are underway, and it will be interesting to see what additional layers of complexity will be added to histone modifications by the modification choice of a single residue.
Many of the enzymes that post-translationally modify histones display not only a high degree of specificity towards a particular site, but also towards the pre-existing modification-state of their substrate. So far the N-terminal tail of H3 has the highest density of post-translational modifications mapped among all histones, and a complex matrix of putative combinations of marks is emerging (FIG. 1B). Methylation on H3-K9, for example, appears to trigger sequential events leading ultimately to transcriptional repression (Wang et al., “Purification and Functional Characterization of a Histone H3-lysine 4-Specific Methyltransferase,” Mol Cell 8:1207-1217 (2001)). At least in vitro, this mark can inhibit acetylation of the H3 tail (on K14, K18, and K23) by HATs (e.g., p300) (Wang et al., “Purification and Functional Characterization of a Histone H3-lysine 4-Specific Methyltransferase,” Mol Cell 8:1207-1217 (2001)), and methylation of H3 on K4 by HMTs (e.g., Set7) (Wang et al., “Purification and Functional Characterization of a Histone H3-lysine 4-Specific Methyltransferase,” Mol Cell 8:1207-1217 (2001)). In contrast, H3-K4 methylation inhibits K9 methylation by Su(var)3-9, but promotes acetylation of H3 by p300 (Wang et al., “Purification and Functional Characterization of a Histone H3-lysine 4-Specific Methyltransferase,” Mol Cell 8:1207-1217 (2001)).
Remarkably, the choice of methylation of H3 on K9 could be dictated by H3-S10 phosphorylation. In mammalian cells, this mark not only inhibits methylation on K9 (Rea et al., “Regulation of Chromatin Structure by Site-specific Histone H3 Methyltransferases,” Nature 406:593-599 (2000)), but also precedes and promotes acetylation on K14 following specific signals (Cheung et al., “Synergistic Coupling of Histone H3 Phosphorylation and Acetylation in Response to Epidermal Growth Factor Stimulation,” Mol Cell 5:905-915 (2000)), see also (Cheung et al., “Signaling to Chromatin Through Histone Modifications,” Cell 103:263-271 (2000)) and references therein). In Saccharomyces cerevisiae, Sfl1 and Gcn5, the enzymes that phosphorylate H3-S10 and acetylate H3-K14, respectively, appear to work synergistically to mediate gene activation (Lo et al., “Snf1—A Histone Kinase That Works in Concert with the Histone Acetyltransferase Gcn5 to Regulate Transcription,” Science 293:1142-1146 (2001)). Moreover, acetylation on H3-K9 and H3-K14 stimulates methylation of H3-K4 by the HMT, MLL (for mixed lineage leukemia protein) (Milne et al., “MLL Targets SET Domain Methyltransferase Activity to Hox gene Promoters,” Mol Cell 10:1107-1117 (2002)). This result is consistent with the enrichment of histones carrying these modifications on HOX gene promoters as shown by ChIP assays (Milne et al., “MLL Targets SET Domain Methyltransferase Activity to Hox gene Promoters,” Mol Cell 10:1107-1117 (2002)). Conversely, methylation on H3-K4 itself can stimulate the subsequent acetylation of H3 (as discussed above). In vitro, further interplay is seen at the level of H3-S10 phosphorylation by the mitotic kinase Ipl1/aurora, which is stimulated when H3-K9 and H3-K14 are acetylated (Rea et al., “Regulation of Chromatin Structure by Site-specific Histone H3 Methyltransferases,” Nature 406:593-599 (2000)).
Additional tail-restricted cross-talk is emerging from studies on modifications of H4 (FIG. 1C). Methylation of H4-R3 by PRMTI, for example, is heavily impaired by acetylation of H4 on K5, K8, K12, and K16 (Wang et al., “Methylation of Histone H4 at Arginine 3 Facilitating Transcriptional Activation by Nuclear Hormone Receptor,” Science 293:853-857 (2001)). In contrast, acetylation of H4 on K8 and K12 by the HAT p300 is elevated after methylation of R3 (Wang et al., “Methylation of Histone H4 at Arginine 3 Facilitating Transcriptional Activation by Nuclear Hormone Receptor,” Science 293:853-857 (2001)). Also, it has been suggested that methylation of K20 and acetylation of K16 are mutually exclusive to each other (Nishioka et al., “PR-Set7 is a Nucleosome-specific Methyltransferase that Modifies Lysine 20 of Histone H4 and is Associated with Silent Chromatin,” Mol Cell 9:1201-1213 (2002)). The local cross-talk situation is likely to be more complicated in vivo, and enzymes that modify the same site might be influenced differently by the modification-state of their substrate.
Perhaps more fascinating than the direct synergism/antagonism or ‘communication’ of adjacent modifications in the same histone tail (‘cis’ effects) is the unexpected discovery that modifications on different histones can affect each other (‘trans’ effects) (Wang et al., “Purification and Functional Characterization of a Histone H3-lysine 4-Specific Methyltransferase,” Mol Cell 8:1207-1217 (2001); Sun et al., “Ubiquitination of Histone H2B Regulates H3 Methylation and Gene Silencing in Yeast,” Nature 418:104-108 (2002); Briggs et al., “Gene Silencing: Trans-histone Regulatory Pathway in Chromatin,” Nature 418:498 (2002); Dover et al., “Methylation of Histone H3 by COMPASS Requires Ubiquitination of Histone H2B by Rad6,” J Biol Chem 277:28368-28371 (2002); and Ng et al., “Ubiquitination of Histone H2B by Rad6 is Required for Efficient Dot1-Mediated Methylation of Histone H3 Lysine 79,” J Biol Chem 277:34655-34657 (2002)). These effects might be restricted to a single nucleosome or might affect larger nucleosomal arrays or domains (FIG. 2). For example, in vitro studies using p300 showed that this HAT acetylates both H3 and H4 especially in nucleosomes where H3 is methylated on K4 (Wang et al., “Purification and Functional Characterization of a Histone H3-lysine 4-Specific Methyltransferase,” Mol Cell 8:1207-1217 (2001)). In contrast, methylation of H3 on K9 significantly inhibits the activity of p300 towards nucleosomal histones, H3 as well as H4 (Wang et al., “Purification and Functional Characterization of a Histone H3-lysine 4-Specific Methyltransferase,” Mol Cell 8:1207-1217 (2001)).
Another intriguing ‘trans’ cross-talk originates from work in Saccharomyces cerevisiae linking ubiquitination of H2B to methylation of H3 (FIG. 2A) (Sun et al., “Ubiquitination of Histone H2B Regulates H3 Methylation and Gene Silencing in Yeast,” Nature 418:104-108 (2002); Briggs et al., “Gene Silencing: Trans-histone Regulatory Pathway in Chromatin,” Nature 418:498 (2002); Dover et al., “Methylation of Histone H3 by COMPASS Requires Ubiquitination of Histone H2B by Rad6,” J Biol Chem 277:28368-28371 (2002); and Ng et al., “Ubiquitination of Histone H2B by Rad6 is Required for efficient Dot1-Mediated Methylation of Histone H3 Lysine 79,” J Biol Chem 277:34655-34657 (2002)). Ubiquitination of H2A and H2B in mammalian cells had been known for a long time (e.g. ubiquitin was discovered on H2A (Goldknopf et al., “Isolation and Characterization of Protein A24, a “Hitone-like” Non-histone Chromosomal Protein,” Journal of Biological Chemistry 250:7182-7187 (1975)), but without an obvious link to protein turnover, the consequences and functions of histone monoubiquitination had been elusive. With the discovery of monoubiquitination of H2B in yeast, genetic studies of histone ubiquitination became possible (Robzyk et al., “Rad6-dependent Ubiquitination of Histone H2B in Yeast,” Science 287:501-504 (2000)). Surprisingly, mutagenesis of either the ubiquitin acceptor site, H2B-K123 (equals human H2B-K120), or disruption of the ubiquitin-conjugating enzyme Rad6/Ubc2 in this organism results in a striking loss of methylation at H3-K4 and H3-K79 (Sun et al., “Ubiquitination of Histone H2B Regulates H3 Methylation and Gene Silencing in Yeast,” Nature 418:104-108 (2002); Briggs et al., “Gene Silencing: Trans-histone Regulatory Pathway in Chromatin,” Nature 418:498 (2002); Dover et al., “Methylation of Histone H3 by COMPASS Requires Ubiquitination of Histone H2B by Rad6,” J Biol Chem 277:28368-28371 (2002); and Ng et al., “Ubiquitination of Histone H2B by Rad6 is Required for efficient Dot1-Mediated Methylation of Histone H3 Lysine 79,” J Biol Chem 277:34655-34657 (2002)). Altogether, these results indicate that ubiquitination of H2B is a prerequisite for methylation of H3 on K4 and K79. On the other hand, abolishment of H3-K4 or H3-K79 methylation has no effect on H2B ubiquitination, suggesting that the cross-talk is unidirectional. This control of a modification pattern in ‘trans’ is site-specific since another site of methylation of H3 in yeast, K36, is not affected (Briggs et al., “Gene Silencing: Trans-histone Regulatory Pathway in Chromatin,” Nature 418:498 (2002)) (note: methylation of H3-K27 has not been detected in budding yeast (Cao et al., “Role of Histone H3 Lysine 27 Methylation in Polycomb-group Silencing,” Science 298:1039-1043 (2002)).
Interestingly, inter-histone cross-talk may not be restricted to a single nucleosome. In yeast, about 5% of H2B is estimated to be ubiquitinated (Sun et al., “Ubiquitination of Histone H2B Regulates H3 Methylation and Gene Silencing in Yeast,” Nature 418:104-108 (2002) and Robzyk et al., “Rad6-dependent Ubiquitination of Histone H2B in Yeast,” Science 287:501-504 (2000)), about 35% of the total H3 pool is thought to be methylated on K4 (Sun et al., “Ubiquitination of Histone H2B Regulates H3 Methylation and Gene Silencing in Yeast,” Nature 418:104-108 (2002)), and 90% of all H3 is methylated on K79 (van Leeuwen et al., “Dot1p Modulates Silencing in Yeast by Methylation of the Nucleosome Core,” Cell 109:745-756 (2002)). Since ubiquitination of H2B appears to be far sub-stochiometric to the methylation of H3, the newly discovered control mechanism might serve as a paradigm for ‘master control switches’ directing the modification pattern of a whole nucleosomal region (FIG. 2B).
Another remarkable feature about this ‘trans-communication’ is the cross-talk between distinct regions of the histone proteins: the N-terminal tail (H3-K4), the histone core region (H3-K79) and the C-terminal tail (H2B-K123) (see FIG. 2A). So far, methylation on H3-K79 is the only known site of modification identified that lies within the nucleosome core domain (see FIG. 1B and (van Leeuwen et al., “Dot1p Modulates Silencing in Yeast by Methylation of the Nucleosome Core,” Cell 109:745-756 (2002) and Ng et al., “Lysine Methylation Within the Globular Domain of Histone H3 by Dot1 is Important for Telomeric Silencing and Sir Protein Association,” Genes Dev 16:1518-1527 (2002)). However, additional sites of modification in the globular region of H3 or other core histones may exist. Genetic studies in Saccharomyces cerevisiae, for example, have identified two patches of sequence in the globular regions of H3 and H4 that are crucial for gene silencing mechanisms and heterochromatin formation (Park et al., “A Core Nucleosome Surface Crucial for Transcriptional Silencing,” Nat Genet 32:273-279 (2002)). In the crystal structure of the nucleosome, these regions are located at the H3/H4 histone-fold motif centered around H3-K79 (see FIGS. 1B and 1C). Whether other, yet unknown, modifications in these patches provide additional cross-talk for the establishment of distinct chromatin readouts is an intriguing possibility.
Besides cross-talk between different covalent modifications, another way of ‘communication’ within the nucleosome core could be disulfide bond-mediated dimerization. It may not be a coincidence that H3 is the only core histone containing a single cysteine (C110), which is conserved in all species except for budding yeast. Formation of a disulfide bond between the two H3 molecules of each nucleosome might place severe conformational restraints on the structure of individual nucleosomes, nucleosomal arrays or chromosomal domains (see FIGS. 1B and 2A for the positioning of C110 within H3 and a nucleosome, respectively). Early pioneering studies using iodoacetamide labeling have indicated that disulfide-linkage of H3 via C110 correlates with transcriptional silencing (Prior et al., “Reversible Changes in Nucleosome Structure and Histone H3 Accessibility in Transcriptionally Active and Inactive States of rDNA Chromatin,” Cell 34:1033-1042 (1983)). Nucleosomes in active regions, in contrast, might be actively maintained in a more reduced, and presumably more open, state. Such reduced regions overlap with hyperacetylated nucleosomes as indicated by mercury-column chromatography (Chen-Cleland et al., Recovery of Transcriptionally Active Chromatin Restriction Fragments by Binding to Organomercurial-agarose Magnetic Beads. A Rapid and Sensitive Method for Monitoring Changes in Higher Order Chromatin Structure During Gene Activation and Repression,” J Biol Chem 268:23409-23416 (1993)).
Singular as well as combinatorial histone modifications obviously impact on chromatin organization and structure. How is a specific modification pattern then translated into changes in genome status and activity? Modifications could directly interfere with the integrity and stability of a single nucleosome or an array of nucleosomes. Bulk acetylation, for example, has been shown to (i) alter the secondary structure of the histone tail, (ii) weaken histone tail-DNA interactions, and (iii) reduce internucleosomal interactions and chromatin folding (see (Annunziato et al., “Role of Histone Acetylation in the Assembly and Modulation of Chromatin Structures,” Gene Expr 9:37-61 (2000)) for references). These effects seem to result directly from changes in the net charge of the histone tails upon acetylation rather than from the presence of the actual mark. Besides biophysical experiments, genetic studies, for example, on the acetylation of the histone variant H2A.Z in Tetrahymena (Ren et al., “Histone H2A.Z Acetylation Modulates an Essential Charge Patch,” Mol Cell 7:1329-1335 (2001)) and of the H4 tail in Saccharomyces cerevisiae (Megee et al., “Genetic Analysis of Histone H4: Essential Role of Lysines Subject to Reversible Acetylation,” Science 247:841-845 (1990)), support such a global readout of this modification via direct effects on nucleosome and chromatin structure (see also (Kristjuhan et al., “Transcriptional Inhibition of Genes With Severe Histone h3 Hypoacetylation in the Coding Region,” Mol Cell 10:925-933 (2002)).
However, other studies have shown that the biological effects of certain distinct marks appear to rely more on specific local binding factors. This docking of effectors to post-translationally modified chromatin is reminiscent of the modular interactions in other signaling pathways (see for example the recruitment of SH2 domains to phospho-tyrosines; for references see (Pawson et al., “Protein-protein Interactions Define Specificity in Signal Transduction,” Genes Dev 14:1027-1047 (2000)). Bromodomains present in several HATs and chromatin remodeling proteins, as well as in the general transcription factor TAF250, bind acetylated lysines (for review see (Zeng et al., “Bromodomain: an Acetyl-lysine Binding Domain,” FEBS Lett 513:124-128 (2002)). Sequential recruitment and anchoring of bromodomain-containing factors and complexes to the promoter region is indeed crucial for the activation of some genes (Agalioti et al., “Deciphering the Transcriptional Histone Acetylation Code for a Human Gene,” Cell 111:381-392 (2002) and Hassan et al., “Function and Selectivity of Bromodomains in Anchoring Chromatin-Modifying Complexes to Promoter Nucleosomes,” Cell 111:369-379 (2002)). Proteins containing certain chromodomains, on the other hand, have been predicted to have affinity for methylated lysines (Jacobs et al., “Structure of HP1 Chromodomain Bound to a Lysine 9-methylated Histone H3 Tail,” Science 295:2080-2083 (2002)). In fact, heterochromatin protein 1 (HP1) can bind to methylated H3-K9 (Jacobs et al., “Specificity of the HP1 Chromo Domain for the Methylated N-terminus of Histone H3,” Embo J 20:5232-5241 (2001); Bannister et al., “Selective Recognition of Methylated Lysine 9 on Histone H3 by the HP1 Chromo Domain CBP/p300 as a Co-factor for the Microphthalmia Transcription Factor,” Nature 410:120-124 (2001); and Lachner et al., “Methylation of Histone H3 Lysine 9 Creates a Binding Site for HP1 Proteins,” Nature 410:116-120 (2001)), and more recent work suggests that the silencing protein Polycomb (Pc) can bind methylated H3-K9 and/or methylated H3-K27 (Cao et al., “Role of Histone H3 Lysine 27 Methylation in Polycomb-group Silencing,” Science 298:1039-1043 (2002); Czermin et al., “Drosophila Enhancer of Zeste/ESC Complexes Have a Histone H3 Methyltransferase Activity that Marks Chromosomal Polycomb Sites,” Cell 111:185-196 (2002); and Kuzmichev et al., “Histone Methyltransferase Activity Associated with a Human Multiprotein Complex Containing the Enhancer of Zeste Protein,” Genes Dev 16 (2002)). It will be interesting to determine if other chromodomain-containing proteins bind yet other sites of lysine methylation in histones, or potentially, in non-histone proteins. Considering the enormous variability of histone modifications, it is likely that a number of other recognition modules still await discovery.
Already, several short clusters of adjacent or closely-spaced modifiable residues are observed in all core histones and well studied ‘hot spots’ of clustered marks punctuate the tails of H3 and H4 (FIG. 3). Whereas in vitro experiments using purified enzymes imply that only a limited number of potential combinations of marks on single histones and possibly nucleosomes might exist (Fischle et al., “Histone and Chromatin Cross-talk,” Curr. Opin. Cell Biol. 15:172-83 (2003)), initial mass spectrometric analysis of histones purified from various cellular sources point to more complex modification patterns in vivo. It seems unlikely that the multiple covalent marks found on histones have evolved independently. The extreme high density and versatility observed might instead serve valuable biological purposes—especially since most sites of post-translational modification are extremely conserved.
The present invention is directed to a novel histone modification biological readout in the form of a binary switch and to uses for that binary switch.